Facile synthesis of human insulin by modification of porcine insulin

ABSTRACT

A method of converting natural porcine insulin to human insulin by reversibly blocking the six carboxyl groups in porcine insulin, digesting the blocked porcine insulin with trypsin to remove the carboxyl terminal octapeptide from the porcine insulin B chain and generating a free carboxyl group in the arginine residue in position 22 of the B chain, and then reversibly blocking all amino groups preferably with BOC azide and coupling the free carboxyl at position 22 to a synthetic octapeptide which corresponds to the sequence at the carboxyl end of the B chain in human insulin, and finally removing all protecting groups from carboxyl and amino and purifying and recovering synthetic human insulin. This method is also susceptible to preparing radioactively labelled human insulin by utilizing radioactive amino acids in the synthesis of the octapeptide grafted or placed on the porcine residue. Further the present method also permits the facile synthesis of insulin analogs, since any selected amino acid sequence may be attached to the carboxyl group at position 22. The above method, in summary, by means of chemical and enzymatic treatment, makes possible the removal quantitatively and selectively of the carboxyl terminal octapeptide from the porcine insulin B chain. This fragment may be replaced by an analogous synthetic human octapeptide to give a protein which is identical to human insulin by a satisfying number of criteria.

United States Patent I 1 Ruttenberg 1451 Sept. 2, 1975 FACILE SYNTHESISOF HUMAN INSULIN BY MODIFICATION OF PORCINE INSULIN Michael A.Ruttenberg, Newton Highlands. Mass.

[75] Inventor:

[73] Assignee: The United States of America as represented by theSecretary of Health, Education. and Welfare, Washington. DC.

221 Filed: Dec. 26, 1972 211 Appl. No.: 318,247

OTHER PUBLICATIONS Prout. Metabolism, 12. 673-675, (1963).

Bromer et al., Biochem. Biophys. Acta, 133, 219-223, (1967). Levy 'etal., Biochem.. 6, 3559-3568, (1967).

Primary E,\'uminer-Elbcrt L. Roberts Assistant Examiner-Reginald J.Suyat [57] ABSTRACT A method of converting natural porcine insulin tohuman insulin by reversibly blocking the six carboxyl groups in porcineinsulin. digesting the blocked porcine insulin with trypsin to removethe earboxyl terminal octapeptide from the porcine insulin B chain andgenerating a free carboxyl group in the arginine residue in position 22of the B chain, and then reversibly blocking all amino groups preferablywith BOC azide and coupling the free carboxyl at position 22 to asynthetic octapeptide which corresponds to the sequence at the carboxylend of the B chain in human insulin, and finally removing all protectinggroups from carboxyl and amino and purifying and recovering synthetichuman insulin. This method is also susceptible to preparingradioactively labelled human insulin by v utilizing radioactive aminoacids in the synthesis of the octapeptide grafted or placed on theporcine residue. Further the present method also permits the facilesynthesis of insulin analogs. since any selected amino acid sequence maybe attached to the carboxyl group at position 22.The above method, insummary, by means of chemical and enzymatic treatment. makes possiblethe removal quantitatively and selectively of the carboxyl terminaloctapeptide from the porcine insulin B chain. This fragment may bereplaced by an analogous synthetic human octapeptide to give a proteinwhich is identical to human insulin by a satisfying number of criteria.

5 Claims. N0 Drawings FACILF. SYNTHESIS OF HUMAN INSULIN BY The completede novo synthesis of insulin has been MODIFICATION OF PORCINE INSU INsuccessfully accomplished in a number .of laboratories as reported, forexample. in Y.-T. Kung. et al, Sci.

Sinica (Peking) l4. 1710(1965). While these chemical present majorbreakthroughs in synthetic protein chemistry. they are in no waypractical for large scale and inexpensive production of insulin. The denovo synthesis of a protein containing 5l amino acid residues is timeconsuming and expensive. In addition. stage where the A and B chains arelinked together by disulfide bridges. In this regard, the presentinvention has a special advantage in that at no stage of the process arethe disulfide bridges between the A and B chain disrupted.

Up to this time. the only practical supply of insulin for treatment ofdiabetes mellitus has been from animal sources. The utilization ofanimal sources. usually beef and pork. has presented a problem ofimmunologic intolerance among treated patients to these non-humanproteins [C. C. Pope. Adv. lmmunol. 5. 209 1966)]. This intolerance maybe a primary cause of the pathological changes that occur among diabeticpatients maintained on insulin therapy for extended periods of time.Therefore, it is the purpose of the present invention to develop insulinidentical in structure to the human protein which could then for thefirst time be made available for such patients. The estimated cost ofthe present synthesized human protein versus the now 30 utilized porcineinsulin is about 2:1. but for sustained therapy the cost differential isbelieved to be out- The present process in outline form is set out belowStep 1. Reversible blocking of all'of the six carboxyl groups in porcineinsulin. These comprise the four y-carboxyl groups from glutamic acidresidues and the two terminal carboxyl groups. The carboxyl groups arepreferably blocked by converting to'the methyl esters and the termreversible is utilized since they are un- Step 2. Digestion of blockedporcine insulin with trypsin. Referring to Table I, it is noted that thealkaline 45 proteinase trypsin scvers the B chain at 22, the arginineposition, and also acts to sever that portion of the chain between (29)lysine and (30) alanine. The activity of trypsin also generates a singlefree carboxyl group at the arginine residue at position 22 of the Bchain.

Step 3. Reversible blocking of all free amino groups preferably witht-butyloxycarbonyl azide (BOC azide) in the manner of Levy andCarpenter. Biochem 6, 3559 Step 4. Coupling of the free carboxyl atposition 22 to a synthetic octapeptide which corresponds to the sequenceat the carboxyl end of the B chain in human in- Step 5. Removal of allprotecting groups of Steps 1 and 3 and purification of the humaninsulin.

In the procedure above, it is noted that the most preferred blockinggroup for the carboxyl as in Step I was by formation of the methyl esterfrom diazomethane and that the most preferred group for blocking of theamino groups was the BOC azide group of Step 3. In both cases. selectionof the blocking agent was made in order to be able to nullify or reversethe protection or blocking in the last stage. which stage comprisesfreeing the active groups and purifying the synthetic human insulin.

EXAMPLE 1 Porcine insulin was obtained from Elanco Products and waspurified by chromatography on a column of carboxymethylcellulose with alinear gradient of volatile pyridine acetate buffer. In a typicalexperiment, 2.7 g. of human insulin were recovered from a startingmaterial of 4.0 g. of purified porcine insulin. The pork insulin wastreated with diazomethane at a pH of 4.6 in a pH stat as described byChibnall. Mangen. and Rees, Biochem. J. 68. 114 (1958). lt was 'foundthat the six carboxyl groups were quantitatively converted to the methylesters with no apparent side reactions. The extent of esterification wasdetermined by microanalysis for methoxyl content.

The porcine insulin methyl ester prepared above was then digested withchymo-trypsin-free trypsin at a pH of 7.5 for hours according to theprocedure essentially as described in Young and Carpenter. J. Biol.Chem.,236, 743 (1961). The digestion by trypsin was quantitative. andwhen monitored in the pH stat, was found to reach completion in 45minutes. As compared with the prior art. the digestion in trypsin wasrapid [Young and Carpenter, J. Biol. Chem. 236, 743 (1961) and Carpenterand Baum, J. Biol. Chem. 237. 409 (1962)]. This rapid digestion it isbelieved may be a consequence of the absence of a negatively chargedgroup normally present at the glutamic acid adjacent to arginine in theB chain. together with the absence of the negative charge at thecarboxyl terminal alanine ad- .jacentto'lysine, as a result of theesterification.

The entire material from the tryptic digestion was applied to a 4 X 100cm column of Sephadex G-75 and eluted with 0.2 M acetic acid. Thedesired product of desoctapeptide insulin pentamethyl ester emerged as asingle peak. Amino acid analysis of this product material is given inTable 11.

4 A' second -p.e'ak';" contained- -the. heptapeptidegly-phe-zphe-tyr-thr pro-lys. as vwell as the alanine methyl ester.These products were separated on Sephadex (3-10 in 0.2 M acetic acidand.th eir composition 7 was confirmed by amino acid analysis. Theproduct material in peak 1 was. isolatedby lyophilization and thentreated with BOC azide asdescribed by Levy andCarpentcr, ante. The BOCazide reacted quantitatively with the two free amino groups in themolecule. It is 10 noted that from the viewpoint of peptide couplingreactions. the only functional group on the molecule at this point isthe terminal carboxyl group at arginine on the B chain. position 22, andtherefore this material was suitable for coupling to the amino terminalgroup on a synthetic human octapeptide.-

EXAMPLE 11 group was removed by catalytic hydrogenation, giving thetripeptide pro-e-BOC-lys-thr methyl ester, which crystallized frommethanol. A pentapeptide CBZ-glyphe-phe-OBz-tyr-OBz-thr was prepared bymeans of a solid phase method [Merrifie1d, J. Am. Chem. Soc. 85.

2149 (1963)]. The appropriate BOC amino acids (Sigma Biochemicals) wereemployed and the pentapeptide hydrazide was obtained by treatment of thepentapeptidyl resin with hydrazine as described by Ohno and Anfinsen, J.Am. Chem. Soc. 89, 5994 The pentapeptide hydrazide above was treatedwith nitrous acid to form the azide, which was then combined with thefree tripeptide pro-e-BOC-lys-thr methyl ester. Hydrogenation of theoctapeptide with palladium catalyst on charcoal gave NH-gly-phe-phe-tyr-thr-pro- TABLE I1.

Amino acid composition of peptides used. Quantitative amino acidanalysis was performed on acid hydrolysates using a Bcckman-Spinco model1208 amino acid analyzer according to the procedure of Spackman. Stein.and Moore. Anal. Chem. 30, 1 1958). Values found are given as ratioswith respect to lysine or arginine.

Chromtltographically Desoctapcptide Synthetic Human ChromatographicallyPure Partially Pure Porcine Insulin Porcine lnsulin octapeptideSynthetic Human lnsulin Amino Acid Expected Found Expected FoundExpected Found Expected Found Lysine 1 1.0 0 0.1 1 1.0 1 l Histidinc 21.8 -2 1.9 2 1.8 Argininc l 1.0 1 1.0 1 1,0 Aspartic acid 3 2.8 3 2.8 37 Thrconinc 2 1.9 l 0.9 2 1.9 3 2,7 Serine 3 2.7 3 2.8 3 2.8 Glutamicacid 7 6.7 7 7.1 7 6,8 Proline 1 1.0 0 0.0 l 1.0 1 1 0 Glycine 4 4.1 33.2 1 1.0 4 42 Alanine 2 2.1 1 1.1 1 1,] Half eystinc 6 a 6 a 6 u Valine4 3,6 4 3.6 4 3.6 lsoleucinc 2 1.7 2 1.7 2 1.8 Lcucine 6 $7 6 5.7 6 5.7Tyrosine 4 3.9 3 3.1 1 0.9 4 3.8 Phenylalanine 3 3.1 1 0.9 2 2.1 3 3.1

"Not determined.

e-BOC-lys-thr methyl ester. the carboxyl terminal sequence of humaninsulin B chain.

EXAMPLE Ill The synthetic octapeptide of Example I] above was thencoupled to the di-BOC-desoetapeptidc insulin pentamethyl ester preparedabove in Example I using dicyclohexyl carbodiimide in tetrahydrofuran at0, with the addition of i I equivalent of N- hydroxysuccinimide.According to the observations of Zimmerman and Anderson, J. Am. Chem.Soc. 89, 7151 (I967), this method produces peptide coupling withvirtually no racemization, and this was found to be the case.

The purified product was subjected to tryptic digestion, after removalof the BOC groups. and quantitative cleavage formed at the newly formedarginyl-glycine bond.

The synthetic human insulin product was proved by chromatography onSephadex G-75, following removal of the BOC groups. The methyl esterswere saponified as described by Chibnall, Mangen, and Rees, Biochem. J.68, I I4 I958 The chromatograph analysis showed the product to beessentially homogeneous, and the material was found to have the correctamino acid composition for human insulin (cf. Table II). Additionally,assay for biological activity utilizing the mouse convulsion techniqueas described by Katsoyannis and Tomctsko. Proc. Natl. Acad. Sci. US. 55,I554 1966), showed that the in vivo activity utilizing a blind assay wasequivalent to that of porcine insulin. As addditional proof, thedigestion of the product with trypsin quantitatively removed thesynthetic octapeptide and thus confirmed its attachment at position 22of the B chain.

EXAMPLE IV In the same manner as Example ll, human octapeptide wasprepared by straightforward condensation of amines. In this example.however, the starting material, threonine methyl ester, was internallylabelled successively with C and H by an injection of the amino acidaccording to the technique set out in Williams and Chase, Methods inImmunology and lmmunochemistry. Volume I, Academic Press, 387389.

For ease of counting and efficiency of operation, the C amino acidinternal labelling was preferred. and it is noted that loss of aminoacid through metabolism and excretion was minimal. The activity of thefinished protein similar to the product obtained in Example III wastested by the thin window technique in a Geiger- Mucllcr counter.

The embodiments of this invention in which an exclusive property orprivilege is claimed are defined as follows:

l. A method of converting natural porcine insulin to human insulin by a.reversibly blocking the six carboxyl groups in porcine insulin byconversion to the methyl esters;

b. digesting the blocked porcine insulin with trypsin to remove thecarboxyl terminal octapeptide from the porcine insulin B chain andgenerating a free carboxyl group in the arginine residue in position 22of the 3 chain;

c. reversibly blocking all amino groups with BOC azide;

d. coupling the free carboxyl at position 22 to a syn thetic octapeptidewhich corresponds to the sequence at the carboxyl end of the B chain inhuman insulin; I

e. removing all protecting groups from carboxyl and amino and purifyingand recovering synthetic human insulin.

2. The method according to claim 1 wherein the amino groups are blockedwith a BOC azide.

3. The method according to claim 1 wherein the six carboxyl groups ofporcine insulin are reversibly blocked by the formation of methyl estersfrom diazomethane. I

4. The method'a'ccording to claim 1 wherein the synthetic octapeptideutilized for coupling agent is radioactively labell ed.

5. The method according to claim 4 wherein the syn thetic octapeptideutilized is radioactively internally labelled.

l 967, pages

1. A METHOD OF CONVERTING NATURAL PORCINE INSULIN TO HUMAN INSULIN BY A.REVERSIBLY BLOCKING THE SIX CARBOXYL GROUPS IN PORCINE INSULIN BYCONVERSION TO THE METHYL ESTERS, B. DIGESTING THE BLOCKED PORCINEINSULIN WITH TRYPSIN TO REMOVE THE CARBOXYL TERMINAL OCTAPEPTIDE FROMTHE PORCINE INSULIN B CHAIN AND GENERATING A FREE CARBOXYL GROUP IN THEARGININE RESIDUE IN POSITION 22 OF THE B CHAIN, C. REVERSIBLY BLOCKINGALL AMINO GROUPS WITH BOC AZIDE, D. COUPLING THE FREE CARBOXYL ATPOSITION 22 TO A SYNTHETIC OCTAPEPTIDE WHICH CORRESPONDS TO THE SEQUENCEAT THE CARBOXYL END OF THE B CHAIN IN HUMAN INSULIN, E. REMOVING ALLPROTECTING GROUPS FROM CARBOXYL AND AMINO AND PURIFYING AND RECOVERINGSYNTHETIC HUMAN INSULIN.
 2. The method according to claim 1 wherein theamino groups are blocked with a BOC azide.
 3. The method according toclaim 1 wherein the six carboxyl groups of porcine insulin arereversibly blocked by the formation of methyl esters from diazomethane.4. The method according to claim 1 wherein the synthetic octapeptideutilized for coupling agent is radioactively labelled.
 5. The methodaccording to claim 4 wherein the synthetic octapeptide utilized isradioactively internally labelled.